CN111235007A - Liquid drop digital PCR system adopting graphene for heating - Google Patents
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- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
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- B01L7/00—Heating or cooling apparatus; Heat insulating devices
- B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
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Abstract
A liquid drop digital PCR system adopting graphene for heating belongs to the technical field of microfluidic chips. The system comprises a graphene heating sheet, a droplet digital PCR microfluidic chip, a temperature measurement and control system, a liquid inlet system and a fluorescence detection image acquisition system. Graphene is used as a heating material and is sealed on an organic glass chip together with a PDMS chip, and PCR amplification reaction is realized when PCR reaction liquid flows through a micro-channel on the PDMS chip. According to the invention, the graphene is adopted as the heating material, and the graphene material has high heat conductivity coefficient, so that the heat conduction performance is good, the temperature uniformity of each circulating temperature zone after heating is good, and the accuracy and the reliability of the PCR system can be improved. Because the resistivity of the graphene material is very small, and the current of a loop is larger under corresponding voltage, the temperature of two temperature zones can be heated to a set temperature within a short time under the same size, PCR reaction liquid finishes amplification when flowing through a micro-channel, and the time required by the whole digital PCR amplification is greatly shortened.
Description
Technical Field
The invention relates to a liquid drop digital PCR system heated by graphene, and belongs to the technical field of microfluidic chips.
Background
As an emerging material, graphene has been widely used in battery electrode materials, semiconductor devices, transparent display panels, sensors, capacitors, transistors, and the like. The connection between the carbon atoms in the graphene is flexible, and when external force is applied to the graphene, the carbon atom surface can be bent and deformed, so that the carbon atoms do not need to be rearranged to adapt to the external force, and the structure is kept stable. The stable lattice structure enables graphene to have excellent thermal conductivity, and the thermal conductivity coefficient of pure defect-free single-layer graphene is as high as 5300W/mK, so that the graphene is the carbon material with the highest thermal conductivity coefficient. While the resistivity is only about
Omega cm, lower than copper or silver, is the material with the smallest resistivity in the world. Meanwhile, in graphene, electrons can be migrated with high efficiency, and the electron mobility at normal temperature exceeds 15000 cm/V · s, while the conventional semiconductor and conductor, such as silicon and copper, far from graphene performs well, and due to the collision of electrons and atoms, the conventional semiconductor and conductor release some energy in the form of heat, and the graphene is different, and the energy of the graphene is not lost, so the graphene has unusual conductivity.
Since graphene is a novel material with a number of outstanding characteristics, it is particularly important to study its specific applications. The graphene material has good heat conductivity, low resistivity and excellent conductivity, so that the graphene material can be used as a heating material and applied to a heating system. The PCR (polymerase chain reaction, abbreviated as PCR) technique is a means for promoting DNA replication using an in vitro enzyme, and can quantitatively detect a target DNA fragment. Over the past thirty years, PCR technology has gone through substantially three major generations: the PCR method comprises three stages of first-generation gel electrophoresis PCR, second-generation Real-time Quantitative PCR (hereinafter referred to as qPCR) and third-generation digital PCR (digital PCR). Absolute quantification remains a challenge for most qPCR assays. Digital PCR is an end-point detection method for distributed targets, and has become more and more promising in the aspect of absolute quantification of rare samples. The efficiency of the digital PCR is mainly reflected in the amplification of a test sample, and the high efficiency of the digital PCR is mainly reflected in that the temperature rise of the digital PCR can be completed in a short time and more products can be obtained as far as possible. A complete digital PCR detection experiment needs to be subjected to multiple annealing and denaturation amplification circulation processes, and the speed of each process needs to be guaranteed, so that the time for the whole digital PCR detection can be effectively shortened. Therefore, temperature accuracy is an important performance indicator in the amplification needs of digital PCR. Not only the temperature rise rate determines the time of the whole digital PCR, but also the detection result can be seriously influenced by larger temperature deviation, so that the non-specific amplification of the experimental sample can be caused, or false positive or false negative can be generated. Therefore, the novel material graphene is adopted as the heating material. Due to the fact that the graphene material is high in heat conductivity coefficient, the heat conducting performance is good, and the heating temperature uniformity is good. Because the resistivity of the graphene material is very small, and the current of a loop is larger under corresponding voltage, the power generated under the same size is larger, the temperature of each circulation temperature zone can be heated to a set temperature in a short time, and the time required by the whole digital PCR amplification is greatly shortened.
The microfluidic chip is a technology for integrating a series of operation units involved in the fields of chemistry, biology and the like into a chip with a square centimeter level or even smaller. Meanwhile, the droplet microfluidic technology has the remarkable advantages of less reagent consumption, mass production of monodisperse droplets, high surface area to volume ratio, independent control of each droplet and the like, and in recent years, the droplet-based microfluidic technology has been developed into a widely-applied multifunctional technology. Compared with the traditional digital PCR, the liquid drop digital PCR combined with the microfluidic chip is greatly improved in the aspects of accuracy and sensitivity, and is simple and convenient to operate and small in sample consumption. Therefore, it has been widely used in the fields related to cell research, disease detection, drug screening, and gene assay.
Disclosure of Invention
The invention aims to provide a liquid drop digital PCR system heated by graphene, which can heat the temperature of each circulation temperature zone to a set temperature in a short time by utilizing the characteristics of good heat-conducting property, good uniformity of the heating temperature and very small resistivity of a graphene material, ensure the uniformity of the temperature distribution of each circulation temperature zone during heating, and simultaneously complete amplification reaction by utilizing micro-channels on a micro-fluidic PDMS chip and adopting a mode of sequentially flowing PCR reaction liquid through a plurality of continuously circulating snake-shaped amplification channels, thereby greatly shortening the time required by the amplification of the whole digital PCR and being capable of improving the accuracy and the sensitivity of the digital PCR.
The technical scheme adopted by the invention is as follows:
a liquid drop digital PCR system adopting graphene for heating comprises a graphene heating sheet, a liquid drop digital PCR microfluidic chip, a liquid inlet system, a temperature measurement and control system and a fluorescence detection image acquisition system, wherein,
the liquid drop digital PCR microfluidic chip comprises a PDMS chip and a glass sheet, wherein a channel is etched on the bottom surface of the PDMS chip, the bottom surface of the PDMS chip is attached to one surface of the glass sheet to form a flow channel, the flow channel comprises a liquid inlet, a liquid inlet flow channel, a reaction flow channel and a liquid outlet, and the liquid inlet, the liquid inlet flow channel, the reaction flow channel and the liquid outlet are sequentially communicated;
the liquid inlet is used for injecting two-phase fluid;
the liquid inlet flow channel is used for converging two-phase fluid input by the liquid inlet system to generate PCR reaction liquid, and the PCR reaction liquid is sequentially circulated in the reaction flow channel in a liquid drop form to complete amplification reaction to generate a PCR amplification product;
the reaction flow channel consists of a plurality of continuous circulating snake-shaped amplification flow channels, and PCR reaction liquid sequentially flows through the plurality of continuous circulating snake-shaped amplification flow channels and completes amplification reaction in the snake-shaped amplification flow channels;
the liquid outlet is used for collecting PCR amplification products after amplification is finished;
the graphene heating sheet corresponds to a reaction flow channel in the droplet digital PCR microfluidic chip and is sealed on the other surface of the glass sheet and used for heating a PCR system;
the temperature measurement and control system is used for controlling and measuring the reaction temperature of the PCR reaction liquid in the droplet digital PCR microfluidic chip;
and the liquid inlet system is used for injecting the two-phase fluid into a flow channel of the droplet digital PCR microfluidic chip and controlling the flow rate of the two-phase fluid.
Further, if the width direction of the droplet digital PCR microfluidic chip is vertical, at least two graphene heating sheets are arranged in parallel up and down.
Furthermore, electrodes are led out from two ends of the graphene heating sheet, the electrodes extend out of the edge of the glass sheet, and a power triode in the temperature measurement and control system is connected with the electrodes in a one-way mode.
Further, the liquid inlet system comprises a syringe pump, and the syringe pump is used for controlling the flow rate of the PCR reaction liquid in the flow channel.
Further, the liquid inlet includes first liquid inlet and second liquid inlet, the feed liquor runner includes first feed liquor runner and second feed liquor runner, first liquid inlet is located the top of second feed liquor runner, the second liquid inlet is located the top of first feed liquor runner, the terminal intercommunication of first feed liquor runner and second feed liquor runner.
Further, the first liquid inlet flow channel and the second liquid inlet flow channel are vertically communicated to form a two-phase flow cross flow channel.
Further, the two-phase fluid is a continuous image fluid and a discrete phase fluid respectively.
The main advantages of the invention are:
(1) the invention adopts the novel material graphene as the heating material, has good heat conduction performance, so that the temperature uniformity of each circulating temperature zone after heating is good, and can be used for improving the accuracy and reliability of the PCR system.
(2) The invention adopts the novel material graphene as a heating material, the resistivity is small, and the current of a loop is larger under corresponding voltage, so that the power generated under the same size is larger, the temperature of two temperature areas can be heated to a set temperature in a short time, and the time required by the whole digital PCR amplification is greatly shortened.
(3) According to the invention, the novel material graphene is adopted as a heating material, the leading-out electrodes at two ends of the graphene are connected into a circuit system, due to collision of electrons and atoms, a traditional semiconductor and a conductor release some energy in a thermal mode, and the energy of the graphene is not lost because the graphene is different.
(4) The liquid drop digital PCR system provided by the invention can greatly shorten the time required by the digital PCR amplification reaction, the PCR amplification reaction is completed in the micro-channel, and the width and the height of the micro-channel are in the micron order, so the time required by the whole digital PCR amplification can be greatly shortened.
(5) The droplet digital PCR system designed by the invention consumes less reagent because of carrying out PCR amplification reaction in the micro-channel, and simultaneously, the PCR reaction solution is amplified in the form of independent droplets, thereby improving the flux of digital PCR.
Drawings
FIG. 1 is a schematic structural diagram of a digital PCR system for liquid drops heated by graphene according to the present invention;
FIG. 2 is a schematic structural diagram of a digital PCR microfluidic chip for liquid drops according to the present invention;
FIG. 3 is a left side view of FIG. 2;
fig. 4 is a two-dimensional plan view of a microfluidic PDMS chip.
The system comprises a temperature measurement and control system 1, a power triode 1-1, a fluorescence detection image acquisition system 2-1, a fluorescence microscope 2-1, a droplet digital PCR microfluidic chip 3, a liquid inlet system 4, a graphene heating sheet 5, a glass sheet 6, a PDMS chip 7, a first liquid inlet flow channel 8-1, a second liquid inlet flow channel 8-2, a reaction flow channel 9, a flow channel 10, a first liquid inlet 11-1, a second liquid inlet 11-2 and a liquid outlet 12.
Detailed Description
The technical solutions in the embodiments of the present invention will be described clearly and completely with reference to the accompanying drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Referring to fig. 1, a droplet digital PCR system heated by graphene comprises a graphene heating plate 5, a droplet digital PCR microfluidic chip 3, a liquid inlet system 4, a temperature measurement and control system 1, and a fluorescence detection image acquisition system 2, wherein,
the liquid drop digital PCR microfluidic chip 3 comprises a PDMS chip 7 and a glass sheet 6, wherein a channel is etched on the bottom surface of the PDMS chip 7, the bottom surface of the PDMS chip 7 is attached to one surface of the glass sheet 6 to form a flow channel 10, the flow channel 10 comprises a liquid inlet, a liquid inlet flow channel, a reaction flow channel 9 and a liquid outlet 12, and the liquid inlet, the liquid inlet flow channel, the reaction flow channel 9 and the liquid outlet 12 are sequentially communicated;
the liquid inlet is used for injecting two-phase fluid;
the liquid inlet flow channel is used for converging the two-phase fluid input by the liquid inlet system 4 to generate PCR reaction liquid, and the PCR reaction liquid is sequentially circulated in the reaction flow channel 9 in the form of liquid drops to complete the amplification reaction to generate PCR amplification products;
the reaction flow channel 9 consists of a plurality of continuous circulating snake-shaped amplification flow channels, and PCR reaction liquid sequentially flows through the plurality of continuous circulating snake-shaped amplification flow channels and completes amplification reaction in the snake-shaped amplification flow channels;
the liquid outlet 12 is used for collecting PCR amplification products after amplification is finished;
the graphene heating plate 5 is sealed on the other side of the glass sheet 6 corresponding to the reaction flow channel 9 in the droplet digital PCR microfluidic chip 3 and used for heating a PCR system, the width of the graphene heating plate 5 is determined by the length of each circulation temperature zone in the digital PCR amplification process, and the width of the graphene material 5 determines the time ratio of PCR reaction liquid passing through each circulation temperature zone. The width of the graphene heating plate 5 and the flow rate of the PCR reaction solution in the flow channel 10 can determine the time required for digital PCR amplification by one cycle, and the time required for the whole digital PCR amplification can be determined by the number of the flow channel cycles in the reaction flow channel 9. The graphene heating sheet 5 is adopted, so that the heat conduction performance is good, the temperature uniformity of each circulating temperature zone after heating is good, and the accuracy and the reliability of a PCR system can be improved; the resistivity is small, so that the temperature of the two temperature zones can be heated up to the set temperature in a shorter time under the same size.
The temperature measurement and control system 1 is used for controlling and measuring the reaction temperature of the PCR reaction liquid in the droplet digital PCR microfluidic chip 3;
and the liquid inlet system 2 is used for injecting the two-phase fluid into the flow channel 10 of the droplet digital PCR microfluidic chip 3 and controlling the flow rate of the two-phase fluid.
Referring to fig. 2 to 3, in the present preferred embodiment, the width direction of the droplet digital PCR microfluidic chip 3 is a vertical direction, and at least two graphene heating plates 5 are arranged in parallel up and down.
Referring to fig. 2, in the preferred embodiment of this section, electrodes are led out from two ends of the graphene heating sheet 5, the electrodes extend out of the edge of the glass sheet 6, and the power transistor 1-1 in the temperature measurement and control system 1 is unidirectionally connected to the electrodes.
Referring to FIG. 1, in the preferred embodiment of this section, the liquid inlet system 4 includes a syringe pump for controlling the flow rate of the PCR solution in the flow channel 10. The flow rate of the PCR reaction solution in the flow channel 10 is determined by the flow rate of the injection pump and the size of the micro flow channel, and the width of the graphene heating sheet 5 and the flow rate of the PCR reaction solution in the flow channel 10 can be adjusted, so that the time required by one cycle of digital PCR amplification is changed.
Referring to FIG. 4, in the preferred embodiment of this section, the liquid inlet comprises a first liquid inlet 11-1 and a second liquid inlet 11-2, the liquid inlet channel comprises a first liquid inlet channel 8-1 and a second liquid inlet channel 8-2, the first liquid inlet 11-1 is located at the beginning of the second liquid inlet channel 8-2, the second liquid inlet 11-2 is located at the beginning of the first liquid inlet channel 8-1, and the ends of the first liquid inlet channel 8-1 and the second liquid inlet channel 8-2 are communicated.
Referring to fig. 2 and 4, in the preferred embodiment of this section, the first liquid inlet flow channel 8-1 and the second liquid inlet flow channel 8-2 are vertically communicated to form a two-phase flow cross flow channel.
In the preferred embodiment of this section, the two-phase fluid is a continuous fluid-like fluid and a discrete phase fluid, respectively.
The working principle of the invention is as follows:
two-phase fluid is pumped into the first liquid inlet 11-1 and the second liquid inlet 11-2 by using an injection pump respectively, the two-phase fluid is converged into liquid drops, namely PCR reaction liquid, at the cross-shaped intersection of the first liquid inlet flow channel 8-1 and the second liquid inlet flow channel 8-2, and the PCR reaction liquid is pumped into the reaction flow channel 9 for amplification reaction. The flow rate of the two-phase flow is controlled by an injection pump to generate stable liquid drops with uniform size and constant speed.
In this embodiment, the graphene heating sheets 5 are provided with two sheets, and are parallelly attached to the glass sheet 6 at positions corresponding to the reaction flow channels 9, so that two constant temperature regions are formed, and the two constant temperature regions are used for annealing and extending the droplet digital PCR microfluidic chip 3. After the droplets are generated, the droplets are sequentially circulated through the two constant temperature regions in the reaction channel 9 to complete the amplification reaction. The width of the graphene heating sheet 5 determines the time ratio of the PCR reaction solution passing through each constant temperature area; the flow rate of the PCR reaction solution in the flow channel 10 is determined by the flow rate of the injection pump and the length, width and height of the micro flow channel; the width of the graphene heating plate 5, the flow rate of the PCR reaction solution in the flow channel 10, and the number of flow channel cycles in the reaction flow channel 9 determine the reaction time.
And after the reaction is finished, collecting the reaction liquid at a reaction liquid outlet, observing the fluorescence number by using a fluorescence microscope 2-1, and obtaining a detection result of the digital PCR through calculation.
Claims (7)
1. A liquid drop digital PCR system heated by graphene is characterized by comprising a graphene heating sheet (5), a liquid drop digital PCR microfluidic chip (3), a liquid inlet system (4), a temperature measurement and control system (1) and a fluorescence detection image acquisition system (2), wherein,
the liquid drop digital PCR microfluidic chip (3) comprises a PDMS chip (7) and a glass sheet (6), wherein a channel is etched on the bottom surface of the PDMS chip (7), the bottom surface of the PDMS chip (7) is attached to one surface of the glass sheet (6) to form a flow channel (10), the flow channel (10) comprises a liquid inlet, a liquid inlet flow channel, a reaction flow channel (9) and a liquid outlet (12), and the liquid inlet, the liquid inlet flow channel, the reaction flow channel (9) and the liquid outlet (12) are sequentially communicated;
the liquid inlet is used for injecting two-phase fluid;
the liquid inlet flow channel is used for converging two-phase fluid input by the liquid inlet system (4) to generate PCR reaction liquid, and the PCR reaction liquid is sequentially circulated in the reaction flow channel (9) in a liquid drop manner to complete amplification reaction to generate PCR amplification products;
the reaction flow channel (9) consists of a plurality of continuous circulating snake-shaped amplification flow channels, and PCR reaction liquid sequentially flows through the plurality of continuous circulating snake-shaped amplification flow channels and completes amplification reaction in the snake-shaped amplification flow channels;
the liquid outlet (12) is used for collecting PCR amplification products after amplification is finished;
the graphene heating sheet (5) corresponds to a reaction flow channel (9) in the droplet digital PCR microfluidic chip (3) and is sealed on the other surface of the glass sheet (6) and used for heating a PCR system;
the temperature measurement and control system (1) is used for controlling and measuring the reaction temperature of the PCR reaction solution in the droplet digital PCR microfluidic chip (3);
the liquid inlet system (2) is used for injecting the two-phase fluid into a flow channel (10) of the droplet digital PCR microfluidic chip (3) and controlling the flow rate of the two-phase fluid.
2. The liquid drop digital PCR system adopting graphene for heating according to claim 1, wherein the width direction of the liquid drop digital PCR microfluidic chip (3) is a vertical direction, and at least two graphene heating sheets (5) are arranged in parallel up and down.
3. The digital PCR system for the liquid drop heated by the graphene as claimed in claim 2, wherein electrodes are led out from two ends of the graphene heating sheet (5), the electrodes extend out of the edge of the glass sheet (6), and a power triode (1-1) in the temperature measurement and control system (1) is connected with the electrodes in a one-way mode.
4. The digital PCR system for liquid drop heated by graphene according to claim 1,
the liquid inlet system (4) comprises a syringe pump, and the syringe pump is used for controlling the flow rate of the PCR reaction liquid in the flow channel (10).
5. The graphene-heated liquid drop digital PCR system according to claim 1, wherein the liquid inlet comprises a first liquid inlet (11-1) and a second liquid inlet (11-2), the liquid inlet channel comprises a first liquid inlet channel (8-1) and a second liquid inlet channel (8-2), the first liquid inlet (11-1) is located at the beginning of the second liquid inlet channel (8-2), the second liquid inlet (11-2) is located at the beginning of the first liquid inlet channel (8-1), and the ends of the first liquid inlet channel (8-1) and the second liquid inlet channel (8-2) are communicated.
6. The digital PCR system for liquid drops heated by graphene according to claim 5, wherein the first liquid inlet flow channel (8-1) and the second liquid inlet flow channel (8-2) are vertically communicated to form a two-phase flow cross flow channel.
7. The graphene-heated droplet digital PCR system according to claim 1, wherein the two-phase fluid is a continuous-phase fluid and a discrete-phase fluid.
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| CN116814414A (en) * | 2023-08-30 | 2023-09-29 | 北京芯畅科技有限公司 | Laser-induced graphene PCR detection device and method |
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